The Random Walk

This is the path to enlightenment for VDS researchers. It may be a direct path or a random walk for you!

These are in general order - but steps can be done in parallel

MILESTONES

Clone your Coding DNA Sequence (CDS) into expression vector

• Primary PCR
• Secondary PCR
• PCR^2
• PCR cleanup, Nanodrop
• Cloning protocol
  • Cut pNIC-Bsa4 (can be done once you have PCR^2 working)
  • Annealing & Transformation
  • Grow up clones
  • DNA sequencing for positives
  • Grow up positives and Midiprep, then archive

Virtual Screening of your target (should be done in parallel with wet lab - i.e. not sequentially)

• Molprobity on structure
• Create Homology Model if needed (run Molprobity on this)
• Pick Control Ligands
• LigPrep Control Ligands
• Set up protein in GOLD & Dock control ligands
• Analyze control docking
• Dock screening libraries of novel compounds
• Order top hits (once you have soluble protein from wet lab)

Express Protein

• Transform plasmid into expression cells
• Express protein
• Harvest & Purify protein through Nickel affinity column and Size Exclusion (FPLC)
• Characterize protein on gel
• Store protein (2 ways)

Enzyme Assays

• Test out if enzyme is active (Vary enzyme)
• Vary substrate assays (determine Km value)

Inhibition Assays

• Test out different concentrations of compounds ordered
• Determine IC50 if it is a good inhibitor

Extra Toppings:
Virtual screening with ICM docking software
Ligand based virtual screening
Fluorescence melt assays
Crystallization trials

Extensions:
carry out MM-PB(GB)SA calculations of Free Energy of Binding to re score top ligands
- use Prime in Maestro Suite from Schrodinger